Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3' end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5'-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) -175 to +76 relative to the SG start site, including the 3' 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5' Deletions of SGP-2 to nt -40 (9 nt beyond the 3' end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5' deletions to nt -26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt -28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt -28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5' end of SG RNA was also required. Thus, the minimal SGP maps from nt -26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt -48 and nt -23 with respect to the SG start site in the RUB genome.